Premature intracellular activation of the digestive enzyme trypsinogen is considered to be the initiating event in pancreatitis. However, the direct consequences of intracellular trypsin activity have not previously been examined. In the current study, a mutant trypsinogen (paired basic amino acid cleaving enzyme (PACE)-trypsinogen), which is activated intracellularly by the endogenous protease PACE, was developed. This new construct allowed for the first time direct examination of the effects of intracellular trypsin on pancreatic acinar cells. We found that PACE-trypsinogen was expressed in the secretory pathway and was activated within acinar cells. Expression of PACE-trypsinogen induced apoptosis of HEK293 cells and pancreatic acinar cells, as indicated by histology, DNA laddering, PARP cleavage, and caspase-3 activation. Cell death was blocked by the trypsin inhibitor Pefabloc but not by the pancaspase inhibitor benzyloxycarbonyl-VAD, indicating that caspase-independent pathways were also involved. However, intracellular trypsin had no significant effect on the activity of the proinflammatory transcription factor NF-B. In contrast, extracellular trypsin caused cell damage and dramatically increased NF-B activity. These data indicate that localization of active trypsin determines its effects on pancreatic acinar cells. This new model will greatly improve our understanding of the role of active trypsin in pancreatitis and its associated inflammatory response."Pancreatitis" is the general term for inflammatory diseases of the pancreas and includes both acute and chronic forms. Pancreatitis results in significant morbidity and mortality, and chronic pancreatitis is associated with an increased risk of pancreatic cancer (1). Unfortunately the mechanisms responsible for the initiation of pancreatitis are not fully understood, which probably explains the persistent lack of causal therapies. More than 100 years ago, Chiari (2) proposed that acute pancreatitis was an autodigestive process, and since that time, the major focus of research on this disease has centered on the role of digestive enzymes (3). Activated digestive enzymes capable of injuring the gland have been detected within the pancreas, pancreatic juice, and plasma in clinical as well as experimental forms of pancreatitis, and the morphological changes that characterize severe pancreatitis indicate that a digestive injury to pancreatic tissue has occurred (4). Furthermore, pretreatment with gabexate mesilate, a serine protease inhibitor, reduces endoscopic retrograde cholangiopancreatography (ERCP)-induced pancreatitis (5). These observations support the suggestion that pancreatitis results from the intrapancreatic activation of digestive enzyme zymogens.Two decades ago, it was observed that serine proteases were activated intracellularly in experimental models of acute pancreatitis (6, 7). These results were taken as a confirmation of the existing paradigm and served to narrow the focus in the field of pancreatitis research to the hypothesis that prema...